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• Integrated genomic analysis for HCC

  Liver cancer is a common malignancy and is the third cause of cancer-related death in the world and hepatocellular carcinoma (HCC) is the major type of liver cancer, accounting for ~90% of cases. Only 20 ~ 30% of patients can benefit from combined immunotherapy and targeted therapy due to the high inter-tumor heterogeneity of HCC. This work aims to identify homogeneous and robust molecular subtypes in HCC based on a large, homogenous and well-annotated cohort. We performed whole genome/exome sequencing (WGS/WES) and/or RNA sequencing (RNA-seq) on 529 HCC from 461 patients collected mainly in France. Based on the great consistence between genomic and transcriptomic data, we identified 9 robust HCC subtypes mainly based on driver mutations. And we further characterized HCC subtypes using genomic, transcriptomic and clinicopathological features. Among 9 subtypes, 5 subtypes belonged to chromosome instable tumors, while 3 subtypes belonged to chromosome stable tumors. In addition, our subtypes were associated with prognosis and we also found different distributions of subtypes across various features, such as etiology and gender. Our study provided a robust molecular classification based on a large HCC dataset from Western country, it improves our understanding of the mechanisms of carcinogenesis and will facilitate development of genome-based precision medicine in HCC.

  Study EGAS00001007957

• Intratumoral Ploidy Dynamics in HCC

  Hepatocellular carcinoma (HCC) ranks as the fourth leading cause of cancer-related deaths. Effective therapies are limited, highlighting the critical need for stratification biomarkers to improve prognosis and predict treatment response. Polyploidization or hyperploidization, an early event observed in many solid cancers, may offer such potential. Here, we developed a new high throughput in situ quantitative approach to assess HCC ploidy profiles. This novel method highlights the presence of both Diploid (DP-) and Hyperploid (HP-) HCCs. RNA-seq profiling classified hyperploid HCCs as belonging to the proliferation subclass, predicting unfavourable clinical outcomes, and revealing a gene expression signature indicative of stemness. Spatially reconstructing nuclear ploidy also revealed DP/HP-HCCs with hyperploid nuclei forming discrete clusters within a predominantly diploid background. Remarkably, these emerging hyperploid foci shared a portion of their transcriptomic programme with fully developed hyperploid HCCs and displayed a signature of genomic instability, including upregulation of multiple DNA repair pathways in response to replication-associated DNA damage. Importantly, spatial transcriptomics of DP/HP HCC demonstrated that hyperploid tumor hepatocytes arise through whole-genome doubling of diploid tumor hepatocytes. Collectively, our findings underscore the prognostic value of nuclear ploidy profiling in HCC and suggest that hyperploid HCCs evolve clonally from intratumor hyperploid foci during tumor progression.

  Study EGAS00001008084

• Constitutional and somatic genomic rearrangements coherently restructure chromosome 21 in acute lymphoblastic leukaemia

  Changes in gene dosage are a major driver of cancer1, engineered from a finite, but increasingly well annotated, repertoire of mutational mechanisms2-6. These processes operate over levels ranging from individual exons to whole chromosomes, often generating correlated copy number alterations across hundreds of linked genes. An example of the latter is the 2% of childhood acute lymphoblastic leukemia (ALL) characterized by recurrent intrachromosomal amplification of megabase regions of chromosome 21 (iAMP21)7,8 To dissect the interplay between mutational processes and selection on this scale, we used genomic, cytogenetic and transcriptional analysis, coupled with novel bioinformatic approaches, to reconstruct the evolution of iAMP21 ALL. We find that individuals born with the rare constitutional Robertsonian translocation between chromosomes 15 and 21, rob(15;21)(q10;q10)c, have ~2700-fold increased risk of developing iAMP21 ALL compared to the general population. In such cases, amplification is initiated by chromothripsis involving both sister chromatids of the dicentric Robertsonian chromosome. In contrast, sporadic iAMP21 is typically initiated by breakage-fusion-bridge (BFB) events, often followed by chromothripsis or other rearrangements. In both sporadic and iAMP21 in rob(15;21)c individuals, the final stages of amplification frequently involve large-scale duplications of the abnormal chromosome. The end-product is a derivative chromosome 21 or a derivative originating from the rob(15;21)c chromosome, der(15;21), respectively, with gene dosage optimised for leukemic potential, showing constrained copy number levels over multiple linked genes. In summary, the constitutional translocation, rob(15;21)c, predisposes to leukemia through a novel mechanism, namely a propensity to undergo chromothripsis, likely related to its dicentric nature. More generally, our data illustrate that several cancer-specific mutational processes, applied sequentially, can co-ordinate to fashion copy number profiles over large genomic scales, incrementally refining the fitness benefits of aggregated gene dosage changes.

  Dataset EGAD00001000658

• Presence of rare potential pathogenic variants in subjects under 65 years old with very severe or fatal COVID‑19

  Rare variants affecting host defense against pathogens could be involved in COVID‑19 severity and may help explain fatal outcomes in young and middle‑aged patients. Our aim was to report the presence of rare genetic variants in certain genes, by using whole exome sequencing, in a selected group of COVID‑19 patients under 65 years who required intubation or resulting in death (n = 44). To this end, different etiopathogenic mechanisms were explored using gene prioritization‑based analysis in which genes involved in immune response, immunodeficiencies or blood coagulation were studied. We detected 44 different variants of interest, in 29 different patients (66%). Some of these variants were previously described as pathogenic and were located in genes mainly involved in immune response. A network analysis, including the 42 genes with candidate variants, showed three main components, consisting of 25 highly interconnected genes related to immune response and two additional networks composed by genes enriched in carbohydrate metabolism and in DNA metabolism and repair processes. In conclusion, we have detected candidate variants that may potentially influence COVID‑19 outcome in our cohort of patients. Further studies are needed to confirm the ultimate role of the genetic variants described in the present study on COVID‑19 severity.

  Study EGAS00001006372

• Comprehensive NGS Profiling to Enable Detection of ALK Gene Rearrangements and MET Amplifications in Non-Small Cell Lung Cancer

  We evaluate the analytical performance of the PGDx™ elio™ tissue complete assay, a 505 gene next-generation sequencing (NGS) tissue-based assay, that has now been FDA-cleared for use by physicians to help guide treatment decisions for cancer patients, using a NSCLC cohort of 38 patients.

  Study EGAS50000000010

• Enhanced resolution profiling in twins reveals differential methylation signatures of Type 2 Diabetes with links to its complications

  We profiled DNA methylation in blood samples from participants of European ancestry from 7 twin cohorts using a methylation sequencing platform targeting regulatory genomic regions encompassing 2,048,698 CpG sites. The results help to improve our understanding of T2D disease pathogenesis and progression and may provide biomarkers for its complications.

  Study EGAS50000000197

• WES to explore mutational landscape of blood and non blood liquids in female patients with metastatic breast cancer.

  Whole Exome Sequencing (WES) to explore mutational landscape of blood and non blood liquids in female patients with metastatic breast cancer. 11 patients. 86 liquid samples, 11 matched normal samples. Median coverage 67X. Illumina paired-end sequencing. Fastq files are provided. WES from Richard et al., Nature Communications, 2025 low-pass Whole Genome Sequencing (lpWGS) to assess the representativeness of seven body liquids from female patients with metastatic breast cancer. 216 liquid samples, 745 solid samples, 20 matched normal samples. 0.5X coverage. lpWGS from Richard et al., Nature Communications, 2025.

  Dataset EGAD50000001852

• Summarized somatic variant calls from CMMRD-associated high-grade gliomas

  This dataset contains summarized somatic variant call data derived from paired tumor–blood whole-exome sequencing of human samples. Access to the dataset is controlled and granted only upon submission and approval of a formal data access request. Requests are reviewed by the Data Access Committee led by the Principal Investigator to ensure compliance with applicable legal, ethical, and data protection requirements. Users must not attempt to re-identify study participants and must comply with all relevant confidentiality and data protection regulations. In accordance with local legal and ethical requirements, individual-level raw sequencing data are not shared.

  Dataset EGAD50000002180

• Tumor Normal Pair for SV mosaic

  Simulated (based on real world data) tumor normal pair designed for benchmarking and optimisation of structural variation callers. The calls are derived from 12 patients in the hartwig database requested by the EUCANCan project. Paired-end sequencing data was imputed into the normal reference data to simulate real world variation like purity as well as technical impact of sequencing technologies. The dataset contains a tumor sequenced to ±90x depth and a normal control sequenced to ±30 x depth of these imputed events. The break junctions of all detected SVs in the original cases are imputed in this data from diagnostic WGS sequencing data.

  Dataset EGAD50000000668

• Cancer Genome Project Exome Sequencing

  Agilent whole exome hybridisation capture was performed on genomic DNA derived from cancer and matched normal DNA from the same patients. Next Generation sequencing performed on the resulting exome libraries and mapped to build 37 of the human reference genome to facilitate the identification of novel cancer genes. Now we aim to re find and validate the findings of those exome libraries using bespoke pulldown methods and sequencing the products.

  Dataset EGAD00001000289

• scRNA-seq to study interactions between HSPCs, BMSCs and immune microenvironment

  Mechanisms of clonal evolution in myeloid neoplasms remain incompletely understood. Darwinian theory predicts that the (micro)environment of clone-propagating stem cells may contribute to clonal selection. Here, we provide data fitting this model, establishing a relationship between stromal niche inflammation, inflammatory stress in HSPCs, clonal resistance and leukemic evolution in human MDS.

  Study EGAS00001008181

• Precursor lesions, clonal architecture and relapse in Wilms nephroblastoma

  Targeted resequencing at high depth (21 genes, 9 chromosomal regions): at least 4 FFPE samples per case and matched germline DNA: * 100 cases with detailed outcome data, including 15 cases with tumour relapse (515 samples) * 40 cases with matched pre-chemotherapy biopsies (240 samples) * 50 nephrogenic rests matched to above cases (50 samples) We expect a proportion (possibly 10%) of cases to be mutationally silent on the above studies, and propose to subsequently carry out integrated whole-genome, methylome and transcriptome studies on matched frozen tissue from these cases

  Dataset EGAD00001003217

• Whole genome sequence of third generation family member (SFHS)

  Previously we performed deep WGS on 6 parents and 13 children from 3 large families from the Scottish Family Health Study to identify de novo mutations. This prelim is cover the additional sequencing of one grandchild from one of these three families. The inclusion of a third generation individual will provide additional experimental validation for the de novo mutations found in the initial trio. As in the previous study, the DNA will be WGS to a depth of approximately 25X to achieve this purpose.These data can only be used for the investigation of the genetic causes of the reported clinical phenotypes in these patients

  Dataset EGAD00001001454

• Transposome Bisulfite Sequencing

  DNA belonging to 16 tumour/normal samples were treated with bisulfite, then up to 5 different bisulfite PCRs were performed in each one of the samples. Amplicons form the same sample were pooled and submitted to sequencing on a MiSeq platform.

  Dataset EGAD00001001028

• Chordoma Sequencing Project Whole Genome

  This is a continuation of the Chordoma Sequencing Project. All cancers arise due to somatically acquired abnormalities in DNA sequence. Systematic sequencing of cancer genomes allows acquisition of complete catalogues of all classes of somatic mutation present in cancer. These mutation catalogues will allow identification of the somatically mutated cancer genes that are operative and characterise patterns of somatic mutation that may reflect previous exogenous and endogenous mutagenic exposures. In this application, we aim to perform whole genome sequencing on 10 chordoma matched genome pairs. RNA Sequencing/Methylation and SNP6 and an additional sequencing of three cancer cell lines will be added to this work.

  Dataset EGAD00001000721

• ENU-LS-411N-TripleTherapy

  BRAF V600E colorectal cancers do not respond to the only currently FDA approved targeted therapy for CRC. There is currently a trial underway in the UK recruiting V600E CRC patients for treatment with a triple therapy combination of Cetuximab, Trametinib and Dabrafenib. We have mutagenized a pool of V600E CRC cell lines and treated with this triple therapy to select out drug resistant clones. We will now sequence these drug resistant clones with the aim of identifying common point mutations engendering resistance to this new therapy.

  Dataset EGAD00001002051

• Single MII oocyte mRNA expression from women

  This dataset contains 14 paired-end FASTQ sequences from mRNA-Seq on single human M-II stage oocytes that were collected from ovarian tissue from unstimulated patients undergoing fertility preservation treatments due to cancer diagnoses, which did not influence ovarian function. Cumulus-oocyte-complexes were matured in vitro according Gruhn et al (Science 365: 1466-1469) and short term flash frozen prior to lysis, RNA extraction, full length cDNA preparation and amplification using the Ultra-low-input SMART-Seq2 v4 kit from Takara Clonetech. Further, these cDNA were used to prepare libraries for sequencing according the Nextera XT DNA library preparation kit from Illumina

  Dataset EGAD00001005971

• Test dataset with ligh-weight files

  This is a test dataset derived from public data of the 1000 Genomes Project. Its purpose is not to allow for any inference about cohort data or results, but to aid bioinformaticians in the technical development and testing of tools, as well as data consumers in learning how to access information. This dataset consists of 3 pairs of light-weight (sliced) files: BAM + BAI, CRAM + CRAI and VCF + TBI. These files can be downloaded directly through the EGA-download-client PyEGA3 (https://github.com/EGA-archive/ega-download-client). For any further questions, please contact the DAC (Helpdesk - email: helpdesk [at] ega-archive [dot] org).

  Dataset EGAD00001009826

• WGS of primary mesothelioma cell lines

  Mesothelioma is an aggressive cancer associated with previous exposure to asbestos and dismal prognosis. Since a pemetrexed/cisplatin combination was introduced for treatment of mesothelioma, no new first- or second-line therapies have been discovered. Thus, to better understand what drives mesothelioma carcinogenesis and to identify potential targets for therapy, in this project we aim at performing WGS analysis of a panel of mesothelioma cells lines.

  Dataset EGAD00001009641

• Embryonic tumour transmission in monozygotic twins

  We have a case of two siblings (twins) who were born with nodular blue rashes over their body. The first twin had a large periorbital subcutaneous mass which was diagnosed as infantile undifferentiated sarcoma. This baby was too systematically unwell for chemotherapy and died at 12 days of age. The second sibling received two cycles of emergency chemotherapy however died due to disease progression and secondary haemorrhage at 2 months of age. Post-mortem normal and tumour samples have been taken. In addition to placental tissue and parental germline DNA, the goal will be to investigate intra-uterine transfer of sarcoma and the phylogenetic relationship of tumours in both twins.

  Dataset EGAD00001015681

• Cellular Indexing of Transcriptomes and Epitopes Sequencing (CITE-Seq) Analysis to Investigate the Impact of Granulocyte-Colony Stimulating Factor on CRISPR/Cas9 Gene Edited Human Hematopoietic Stem Cell Function

  Our research using in-vivo preclinical xenograft mouse models demonstrated that G-CSF impedes engraftment of CRISPR-Cas9 gene edited human hematopoietic stem cells (HSCs). In this CITE-seq study, we sought to investigate the mechanisms by which G-CSF negatively impacts long-term repopulating functions of CRISPR-Cas9 gene edited human HSCs. Accordingly, human CD34+ cells were obtained from a healthy volunteer and edited with Cas9 alone (Cas9 group), Cas9+AAVS1-targeting gRNA (RNP group), or Cas9+AAVS1-targeting gRNA+GSE56 (RNP/GSE56 group). Then, these edited cells were transplanted into busulfan-conditioned NSG mice, and these mice subsequently received 3 days of phosphate buffered saline (PBS) or G-CSF. Each mouse was subsequently euthanized and harvested for bone marrow cells and human CD34+cells were isolated using immunomagnetic beads. Isolated CD34+ cells from a total of 6 mice were individually stained with Total seq B CITE-seq antibodies (CD38, CD45RA, CD90, CD49f) + hashtag oligo antibodies 1, 2, 3, 4, 5 and 6. Fluorescence activated cell sorting (FACS) was used to purify ~2,500 cells from each group that were subsequently pooled for CITE-seq library preparation and sequencing. Seurat was used to demultiplex data into 6 individual groups and differential gene expression analysis was performed to evaluate the impact of G-CSF within Cas9, RNP and RNP/GSE56 groups.

  Study phs003277

• MP2PRT: Comprehensive Genomic Profiling to Identify Alterations Associated with Relapse for NCI Standard Risk (SR) B-Lineage ALL and NCI High Risk (HR) B-Lineage ALL with Favorable Genetic Features

  The Molecular Profiling to Predict Response to Treatment (MP2PRT) program is part of the NCI's Cancer Moonshot Initiative. The aim of this program is the retrospective characterization and analysis of biospecimens collected from completed NCI-sponsored trials of the National Clinical Trials Network and the NCI Community Oncology Research Program. This study, titled "Comprehensive Genomic Profiling to Identify Alterations Associated with Relapse for NCI Standard Risk (SR) B-Lineage ALL and NCI High Risk (HR) B-Lineage ALL with Favorable Genetic Features," is one of the selected studies under this program. For this study, genomic characterization (WGS 30X, WXS, Total RNAseq) was performed on NCI Standard Risk B-lineage ALL patients who relapse and subsets of NCI High Risk B-lineage ALL patients who relapse as well as two non-relapse control cases for each relapse case. It was hypothesized that the comprehensive characterization of these sample sets will allow the identification of somatic genetic features that will facilitate early intervention to improve cure rates for those enrolled on future trials. Data were generated and genetic subtype was integrated with clinical variables, including age, presenting white blood cell count, sex, race, and levels of minimal residual disease at the end of induction. Additional secondary alterations were identified and included if they occurred in more than 10 patients. The study has been completed and a manuscript has been prepared.

  Study phs002005

• Genetic Basis for Clinical Response to CTLA-4 Blockade in Melanoma

  Immune checkpoint inhibitors are effective cancer treatments, but molecular determinants of clinical benefit are unknown. Ipilimumab and tremelimumab are antibodies against cytotoxic T-lymphocyte antigen 4 (CTLA-4). Anti-CTLA-4 treatment prolongs overall survival in patients with melanoma. CTLA-4 blockade activates T cells and enables them to destroy tumor cells. We obtained tumor tissue from patients with melanoma who were treated with ipilimumab or tremelimumab. Whole-exome sequencing was performed on tumors and matched blood samples. Somatic mutations and candidate neoantigens generated from these mutations were characterized. Neoantigen peptides were tested for the ability to activate lymphocytes from ipilimumab-treated patients. Malignant melanoma exomes from 64 patients treated with CTLA-4 blockade were characterized with the use of massively parallel sequencing. A discovery set consisted of 11 patients who derived a long-term clinical benefit and 14 patients who derived a minimal benefit or no benefit. Mutational load was associated with the degree of clinical benefit (P = 0.01) but alone was not sufficient to predict benefit. Using genomewide somatic neoepitope analysis and patient-specific HLA typing, we identified candidate tumor neoantigens for each patient. We elucidated a neo-antigen landscape that is specifically present in tumors with a strong response to CTLA-4 blockade. We validated this signature in a second set of 39 patients with melanoma who were treated with anti-CTLA-4 antibodies. Predicted neoantigens activated T cells from the patients treated with ipilimumab.

  Study phs001041

• Machine learning to detect the SINEs of cancer

  We previously described an approach called RealSeqS to evaluate aneuploidy in plasma cell-free DNA (cfDNA) through the amplification of ~350,000 repeated elements with a single primer. We hypothesized that an unbiased evaluation of the large amount of sequencing data obtained with RealSeqS might reveal other differences between plasma samples from patients with and without cancer. This hypothesis was tested through the development of a novel machine-learning approach called Alu Profile Learning Using Sequencing (A-PLUS) and its application to samples from 5108 individuals, 2037 with cancer and the remainder without cancer. Samples from cancer patients and controls were pre-specified into four cohorts used for: 1) model training, 2) analyte integration and threshold determination, 3) validation, and 4) reproducibility. A-PLUS alone provided a sensitivity of 40.5% across 11 different cancer types in the Validation Cohort, at a specificity of 98.5%. Combining A-PLUS with aneuploidy and 8 common protein biomarkers detected 51% of 1167 cancers at 98.9% specificity. We found that part of the power of A-PLUS could be ascribed to a single feature – the global reduction of AluS sub-family elements in the circulating DNA of cancer patients. We confirmed this reduction through the analysis of another independent dataset obtained with a very different approach (whole genome sequencing). The evaluation of Alu elements therefore has the potential to enhance the performance of several methods designed for the earlier detection of cancer.

  Study EGAS00001007169

• Homozygous inactivation of CHEK2 is linked to a familial case of multiple primary lung cancer with accompanying cancers in other organs

  In clinical practice, there are a number of cancer patients with clear family histories, but not belonging to existing familial malignancies. Recent advances in genomic technologies have enhanced the possibility of identifying causative genes in such cases. Two siblings, an elder sister and a younger brother, were found to have multiple primary lung cancers at the age of 60. The former subsequently developed breast cancer, and had a history of myoma uteri. The latter had initially developed prostate cancer at the age of 59, and had a history of colon cancer. SNP genotyping revealed that approximately 10% of the genomes were homozygous in both patients. Exome sequencing revealed non-synonymous mutations in four genes in the runs of homozygosity: CHEK2, FCGRT, INPP5J, and SFI1. Evolutionary conservation of primary protein structures suggested the functional importance of the CHEK2 mutation, R474C. This mutation altered the tertiary structure of CHK2 by disrupting the salt bridge between R474 and E394. No such structural changes were observed with the other three mutated genes. Subsequent cell-based transfection analysis revealed that CHK2 R474C was unstable and scarcely activated. We concluded that CHEK2 is a causative gene in this case of familial cancer. Although homozygous inactivation of CHEK2 in mice led to cancers in multiple organs, accumulation of additional human cases is needed to establish its pathogenic role in humans.

  Study JGAS000057

• HTAN MCL Pre-Cancer Atlas Pilot Project - Targeted Sequencing Development Study

  The HTAN-MCL Pre-Cancer Atlas Pilot Project (PCAPP) is the result of a collaboration between the seven members of the MCL consortium. Across four organ types, PCAPP's goal is to collect and profile pre-malignant lesions for gene expression, DNA mutations, single-cell gene expression and immune-environment. Most PML are small in size and only available come from formalin fixed paraffin embedded archived tissue. The primary goal of PCAPP is to 1) understand the logistical challenges of PML specimen collection, 2) document technical limitations of the assays that are specific to the PML and 3) overcome them to support the generation of a more comprehensive Pre-Cancer Atlas in the future. The current upload provides RNA and DNA sequencing from participants with DCIS who were studied at the University of San Diego and the University of Vermont. Description of the overall study: A. Background/Significance One of the critical barriers to developing new approaches for cancer detection and prevention is the lack of understanding of the key molecular and cellular changes that cause cancer initiation and progression. Unlike the extensive work that has been done profiling advanced stage tumors, few studies have comprehensively profiled the molecular alterations found in precancerous tissues. Premalignant lesions are currently characterized by histologic changes that precede the development of invasive carcinoma1,2.These lesions can often be identified in regions surrounding an invasive tumor or in biopsies taken from patients undergoing diagnostic evaluation for suspicion of cancer. Currently, limited metrics exist to identify lesions that will likely progress to carcinoma and require intervention from those that will naturally regress or remain stable3,4. Characterization of the molecular alterations in premalignant lesions and the corresponding changes in the microenvironment would hasten the development of biomarkers for early detection and risk stratification as well as suggest preventive interventions to reverse or delay the development of cancer. Our pilot study will establish the feasibility of transcriptomic, genomic and immune profiling of FFPE premalignant lesions from multiple organ sites, collected and profiled with uniform SOPs across multiple institutions within the MCL consortium. We will characterize the molecular alterations in precancerous lesions and the corresponding microenvironment in four major organ sites, in order to uncover the molecular and cellular determinants of premalignancy, and establish standardized sequencing and immunohistochemistry protocols on FFPE precancerous tissue. We will also evaluate the technical feasibility of single nuclei sequencing of small FFPE pre-cancer lesions. Successful completion of the proposed pilot study will set the stage for expansion and development of a comprehensive Pre-Cancer Atlas (PCA) as part of the NCI's moonshot.B. Specific Aims Aim 1: Collect premalignant lesions (PML) and their associated microenvironment via LCM from FFPE tissue across four organ sites (breast, lung, pancreas & prostate). Aim 2: Perform bulk RNA and DNA seq on premalignant FFPE samples (and flash frozen tissue where available) and compare the genomic/transcriptomic alterations within and across organ sites. C. ApproachAim 1: Collect premalignant lesions (PML) and their associated microenvironment via LCM from FFPE tissue across four organ sites (breast, lung, pancreas & prostate). MethodsI. Patient Population/Sample Collection: Overview of the sites collecting PML tissue from the respective organs is provided in Table 1 and a full description of the biospecimens to be obtained is described in detail for each organ type below. Table 1. Breakdown of cohort by tissue type and collection site.Organ siteBreastLungPancreasProstateType of PMLDCISAAH, Squamous Dysplasia/CISIPMNsPINCollection of PatientsUCSF/UCSDUVMBU*/UCLAVanderbilt/MoffittMDACC*JHUStanford*# of Patients201920 (10 of each type)20 (10 of each type)242020Total patients per Organ39402440Note: single nuclei/cell RNA-Seq will be performed on 4-5 FFPE samples from each of the organ types 1. DCIS lesions from breast tissue: DCIS lesions will be collected from 39 patients (20 from UCSF/UCSD & 19 from UVM) with primary low or high-grade DCIS diagnosed from a breast core biopsy. Subsequent resected lumpectomy or mastectomy tissues will be prospectively sampled in the vicinity of the prior biopsy site using multiple approaches: 1) Live cells (heterogeneous mix) will be obtained as a cell scrape slurry from the lesion surface or by fine needle aspirate (FNA); 2) For a subset of specimens where size is sufficient, a block of breast tissue with DCIS will be fresh-frozen; 3) The remainder of the specimen will be taken for routine formalin-fixation and paraffin-embedding (FFPE). The FFPE sample will be annotated to identify the matched FFPE tissue block adjacent to the fresh-frozen sample and will be sectioned for use in bulk and single nuclei sequencing . We will dissect DCIS, adjacent normal and when available, associated carcinoma. In addition, when possible, normal tissue will be collected from a tissue block lacking lesions as well as collection of blood. A subset of patients (n = 5 | FFPE, flash frozen and fresh) will be sent to the Broad Institute for single nuclei/cell sequencing.2. AAH and squamous dysplastic/CIS lesions from airway and lung tissue: For squamous cell lung cancer, we will collect endobronchial biopsies from abnormal airway regions identified on autofluroscence bronchoscopy or identify PMLs in the margins of resected lung tissue. We will study 20 patients (5 each from BU/UCLA/Vanderbilt/Moffitt) with pre-invasive squamous lesions (moderate-severe dysplasia or carcinoma in situ (CIS)) identified on pathologic examination. LCM of the premalignant region and adjacent normal epithelium will be performed as well as the invasive tumor for those collected from the resection margin (n=5 from UCLA). On a subset of lesions collected at bronchoscopy (n=5), we will collect additional biopsies that will be flash frozen and fresh for single nuclei and cell sequencing, respectively, performed at the Broad Institute. In parallel to the work at the Broad, BU will perform single cell RNA-seq on these freshly cell sorted tissues (n = 5). Blood will be collected on all patients for genomic studies. For lung adenocarcinoma, we will collect resected FFPE lung tissues from 20 patients (10 from UCLA and 10 from Vanderbilt/Moffitt) with early stage lung adenocarcinoma that harbor atypical adenomatous (AAH) premalignant lesions in the resection margin. We will LCM multiple AAH regions (3-5 per patient) as well as adjacent regions of normal epithelium and invasive adenocarcinoma. In addition, blood will be collected on all patients for genomic studies. 3. IPMNs from pancreatic tissue: For pancreatic cancer PML, we will collect low and high grade lesions from 24 patients representing macroscopic Intraductal Papillary Mucinous Neoplasms (IPMN) (n=24) from surgically resected specimens along with blood samples. Archival FFPE specimens of microscopic PanIN lesions, occurring multi-focally adjacent to invasive PDAC, and archival IPMN lesions (with or without associated invasive cancer), along with the adjacent normal tissue, will undergo LCM and utilized for bulk DNA and RNA sequencing. If matched frozen tissues are available for a subset of these FFPE samples, we will bank for comparison of profiles. Because IPMNs are macroscopic lesions, they provide an opportunity for obtaining the samples fresh and therefore can be used for single cell sequencing (in contrast to PanINs). Therefore, 5 freshly obtained IPMNs will be used for the single cell RNA sequencing studies performed at both the Broad Institute and MDACC, and the matched FFPE and/or frozen sections from these lesions (obtained from the adjacent PML) will be sent to Broad Institute as a pilot to assess "single nuclei" RNA sequencing.4. PINs from prostate tissue: For prostate cancer PML, there will be 40 samples of Prostatic Intraepithelial Neoplasia (PIN) collected between the Stanford and JHU sites (20 cases per site). At the Stanford site, 20 prostate specimens detected by PSA screening who have/will undergo surgery (radical prostatectomy) for clinically localized disease will make up the final cohort. The age range of the participants would be 40-75, and we anticipate that 18 will be Caucasian, 1 Asian and 1 Latino or African American based on the practice demographics practice at Stanford. Clinical and MRI data will also be collected for these samples. We will collect low grade (e.g. Gleason score of 6/Grade group 1; n=10) and high grade (Gleason score 4+3=7 or higher/Grade group 3 or higher; n=10) PINs from FFPE samples that have prostate carcinoma. In addition to obtaining LCM archival samples of low and high grade PIN, we will also obtain normal prostatic epithelial from the peripheral, central and transition zones as well as multiple samples of prostate carcinoma in order to obtain the spectrum of Gleason grades in the carcinoma as needed. LCM samples will be used for bulk DNA and RNA sequencing. In addition, single cells will be dissected from FFPE samples to prepare single cell RNA seq libraries using techniques developed at Stanford, and FFPE tissue will be sent to the Broad for single nuclei sequencing. When available, flash frozen and fresh samples from these prostates will be archived and prepared for single nuclei and cell sequencing, respectively, at the Broad Institute and at Stanford (single cell only). JHU will also capture 10 cases (5 grade group 1 and 5 grade group 2) of high grade PIN, normal and invasive adenocarcinoma using frozen sections from fresh frozen tissues. When possible these will be from the same patients as the FFPE samples. Since frozen sections can be quite challenging to morphologically determine high grade PIN from normal epithelium, for these samples we will perform a number of additional tissue-based characterizations. These will include a multicolor combined basal cells (p63 and CK903) and PIN/carcinoma markers (AMACR) referred to in the cocktail as "PIN4", c-MYC (referred to as MYC) protein5, by IHC and mRNA by in situ hybridization (AM De Marzo, Q Zheng unpublished observations), telomere length by in situ hybridization6 and the 5'ETS/45S rRNA7. For these slides, the whole slides will be scanned with a Hammamatsu Nanozoomer with a 40x objective and regions of interest will be annotated as a guide for LCM.II. Laser-capture microdissection (LCM): FFPE tissue blocks will be sectioned at 7μm thickness and serial sections will be stained with H&E. LCM will be performed utilizing standard LCM systems, such as Leica LMD7000 and ArcturusXT at each site. Regions of premalignancy will be dissected and RNA/DNA will be extracted from microdissected cells using the Qiagen All Prep DNA/RNA FFPE Kit. Aim 2: Perform bulk RNA and DNA seq on premalignant FFPE samples and compare the genomic/transcriptomic alterations within and across organ sites. Rationale: There have been limited studies characterizing the genomic and transcriptomic landscape of premalignant lesions associated with breast, pancreatic, lung or prostate cancers. Characterizing the molecular determinants of premalignant disease that are unique and shared across multiple organs will enable new candidate biomarkers for early detection and novel therapeutic strategies for early intervention. MethodsBulk RNA-seq of LCM FFPE tissue: All participating sites will perform bulk RNA-seq in accordance with SOPs developed at BU. In brief, total RNA will be isolated from LCM'd lesion and associated microenvironment tissue using the Qiagen All Prep DNA/RNA FFPE Kit and quality will be assessed with the Agilent Bioanalyzer. Libraries will be generated with the Illumina TruSeq Access kit (for FFPE samples). They will be sequenced on the Illumina HiSeq2500 with 75base-pair paired-end reads. Quality of FASTQ files will be assessed with FastQC. Reads will be aligned to the human genome with STAR and gene-level and isoform-level expression will be quantified with RSEM. Splice junction saturation, transcript integrity, and biotype distributions will be calculated for each sample with RSeQC. DESeq2 and EdgeR will be used to identify associations between gene expression profiles and clinical variables while controlling for confounding covariates. BU will serve as an RNA-seq Core to assess reproducibility of FFPE RNA-seq methods across sites. We will perform RNA-seq according to the SOP listed above on a subset of samples for each organ type (total n ~ 20). Bulk seq of DNA from FFPE tissue: All participating sites will perform targeted or whole exome-seq (WES) in accordance with SOPs. In brief, DNA from laser captured material will be isolated using the Qiagen All Prep DNA/RNA FFPE Kit and undergo stringent quality control to ensure high quality input material for genomic profiling. Purified DNA (ideally 100-200 ng) will be used for library preparation and amplification, followed by next generation sequencing using standard protocols distributed by CDMG. Exome-seq methods are considered standardized, thus we will not need a DNA-seq Core to assess reproducibility across sites. We anticipate local centers will use Illumina paired end reads, following the following general approach. 1) DNA library preparation: Paired-end libraries will be prepared following the manufacturer's protocols (Illumina and Agilent), fragmented to 150-200 bp 2) Capture of targeted exome: Whole exome capture will be carried out using the protocol for Agilent's SureSelect Human All Exon kit. Purified capture products will be amplified using the SureSelect GA PCR primers (Agilent) for 12 cycles. 3) Sequencing will be carried out for the captured libraries using at least 100 bp paired-end reads. To achieve high level sensitivity and accuracy for detecting all the mutations in the whole exome, each sample will be sequenced at 200X mean depth. 4) Read mapping and alignment and variant analysis: Sequence short reads will be aligned to a reference genome (NCBI human genome assembly build 38) using BWA-MEM. Local realignment of aligned reads will be performed using Genome Analysis Toolkit (GATK).Data QC: To ensure scientific rigor and consistency among sites in RNA and DNA processing we will include a preliminary analysis of steps in processing and analysis. Protocols for extraction of high quality RNA and DNA from formalin fixed paraffin embedded (FFPE) tissues, which will be used extensively in these studies continue to improve and may have variable implementation among the sites participating in this study. To evaluate consistency of preliminary steps in processing and downstream analyses, we will initially distribute slides from one large FFPE fixed cancer of origin from prostate, breast, lung and pancreatic cancer. Analysis of these samples will allow us to review the DNA and RNA characteristics (yield, purity and strand length) among sites. Downstream analysis of these same samples will also allow us to compare among sites the consistency of variant calls among centers. We will be able to identify if there are some times of calls (such as small insertion deletions) that are more variable among centers versus other types of calls (such as relative gene expression or single base pair substitutions) that we expect to be less variable and to characterize the reliability of findings across sites. We are also including a 5% blind duplicate analysis of RNA sequencing. Samples will be analysed by the participating genomics cores without knowledge of the phenotype. RNA seq and CNA analyses are normalized for batch effects. We will also compare the observed sex to the self-reported sex as based on RNA profiles and exome sequencing of X chromosome genes as another check for processing accuracy and sample management. D. References 1. Wacholder, S. Precursors in Cancer Epidemiology: Aligning Definition and Function. Cancer Epidemiol. Prev. Biomark. 22, 521-527 (2013). PMID: 23549395.2. Berman, J. J. Precancer: The Beginning and the End of Cancer. (Jones & Bartlett Learning, 2011).3. Nasiell, K., Nasiell, M. & Vaćlavinková, V. Behavior of moderate cervical dysplasia during long-term follow-up. Obstet. Gynecol. 61, 609-614 (1983). PMID: 6835614.4. Merrick, D. T. et al. Persistence of Bronchial Dysplasia Is Associated with Development of Invasive Squamous Cell Carcinoma. Cancer Prev. Res. (Phila. Pa.) 9, 96-104 (2016). PMID: 26542061.5. Gurel, B. et al. Nuclear MYC protein overexpression is an early alteration in human prostate carcinogenesis. Mod. Pathol. Off. J. U. S. Can. Acad. Pathol. Inc 21, 1156-1167 (2008). PMID: 18567993.6. Meeker, A. K. et al. Telomere shortening is an early somatic DNA alteration in human prostate tumorigenesis. Cancer Res. 62, 6405-6409 (2002). PMID: 12438224.7. Guner, G. et al. Novel Assay to Detect RNA Polymerase I Activity In Vivo. Mol. Cancer Res. MCR 15, 577-584 (2017). PMID: 28119429.

  Study phs002225

• A Genome-Wide Association Study in Breast Cancer Patients from the Prospectively Randomized SUCCESS Trial

  The SUCCESS-A Study (http://www.success-studie.de) is a randomized Phase III study of response to treatment of early primary breast cancer with adjuvant therapy after surgical resection. The primary outcome is disease-free survival and secondary outcomes are overall survival and toxicity. The study design includes comparison of two pairs of treatment regimes performed in sequence. The first randomization contrasts treatment with and without Gemcitabine (3 cycles of Epirubicin-Fluorouracil-Cyclophosphamide(FEC)-chemotherapy, followed by 3 cycles of Docetaxel(D)-chemotherapy versus 3 cycles of Epirubicin-Fluorouracil-Cyclophosphamide(FEC), followed by 3 cycles of Gemcitabine-Docetaxel(DG)-chemotherapy). The second randomization contrasts treatment with Zoledronate for two versus five years. Patients were recruited from 2005 to 2007 and a total of 3754 patients were included from 250 study sites across Germany. Genome-wide SNP microarray data and phenotype outcomes for the first phase of the study (treatment with and without Gemcitabine) are included in the GARNET (Genomics and Randomized Trials Network) pharmacogenomic study in this dbGaP submission. (The Zoledronate treatments are still underway). SUCCESS-A is a an open-label, multicenter, 2x2 factorial design, randomized controlled, Phase III study comparing the disease free survival after randomization in patients treated with 3 cycles of Epirubicin-Fluorouracil-Cyclophosphamide(FEC)-chemotherapy, followed by 3 cycles of Docetaxel(D)-chemotherapy versus 3 cycles of Epirubicin-Fluorouracil-Cyclophosphamide(FEC), followed by 3 cycles of Gemcitabine-Docetaxel(DG)-chemotherapy, and to compare the disease free survival after randomization in patients treated with 2 years of Zoledronate versus 5 years of Zoledronate in patients with early primary breast cancer. Patients were required to have histopathological proof of axillary lymph node metastases (pN1-3) or high risk node negative, defined as: pT>2 or histopathological grade 3, or age , ≤ 35 or negative hormone receptor, but were not allowed to have evidence of distant disease. Patients were entered into the study no later than 6 weeks after complete resection of the primary tumor. No antineoplastic treatment other than surgical treatment, the defined cytotoxic and endocrine treatment, and radiotherapy was allowed prior to study entry and during the course of the study. After surgery, leading to complete resection of the invasive and intraductal components of the primary tumor, patients were randomized to one of the following two treatments for the first phase of the project (which is included in this GARNET pharmacogenomics study): AA: 3 cycles of 5-Fluorouracil 500 mg/m² i.v. body surface area and Epirubicin 100 mg/m² i.v. and Cyclophosphamide 500 mg/m² i.v., (FEC100), each administered on day 1, repeated on day 22, subsequently followed by 3 cycles of Docetaxel 75 mg/m² body surface area i.v. (D), and Gemcitabine 1000 mg/m² i.v. (30 min infusion) (G), administered on day 1, followed by Gemcitabine 1000 mg/m² i.v. (30 min infusion) on day 8, repeated on day22 AB: 3 cycles of 5-Fluorouracil 500 mg/m² i.v. body surface area and Epirubicin 100 mg/m² i.v. and Cyclophosphamide 500 mg/m² i.v., (FEC100), each administered on day 1, repeated on day 22, subsequently followed by 3 cycles of Docetaxel 100 mg/m² body surface area i.v. (D), administered on day 1, repeated on day 22 A second randomization was made for assigning patients to one of the following two subsequent treatments for the second phase of the project (NOT included in the pharmacogenetic study in this dbGaP submission): BA: Zoledronic acid 4 mg i.v., every 3 months for the duration of two years, subsequently followed by zoledronic acid 4 mg i.v., every 6 months for the duration of additional three years BB: Zoledronic acid 4 mg i.v., every 3 months for the duration of two years Patients with positive hormone receptor status (≥10% positively stained cells for estrogen and/or progesterone) of the primary tumor were recommended to receive Tamoxifen treatment 20 mg p.o. per day for 2 years, after the end of chemotherapy . Subsequent to chemotherapy, postmenopausal patients with positive hormone receptor status were recommended to be treated with Anastrozole (Arimidex®) 1 mg p.o. for additional 3 years, premenopausal patients will continue Tamoxifen treatment for those additional 3 years. In addition to tamoxifen, all patients with positive hormone receptor status of the primary tumor and under the age of 40 or restart of menstrual bleeding within 6 months after the completion of cytostatic treatment or with premenopausal hormone levels as defined below were recommended to receive Goserelin (Zoladex®) 3.6 mg subcutaneously every 4 weeks over a period of 2 years following chemotherapy. Premenopausal endocrine status will be assumed, if the following serum levels are met: LH < 20 mIE/ml, FSH < 20 mIE/ml and E2 > 20 pg/ml. Endocrine therapy will start after the end of chemotherapy. All patients with breast conserving therapy or more than 3 axillary lymph node metastases or in the following cases after mastectomy will receive adjuvant radiotherapy: T3/T4-carcinoma, T2-carcinoma > 3 cm, multicentric tumor growth, lymphangiosis carcinomatosa or vessel involvement, involvement of the pectoralis fascia or a safety margin < 5 mm. Radiotherapy was usually performed after the chemotherapy and parallel to the beginning of the zoledronic acid therapy. Initially, DNA was extracted for performing SNP microarray assays using blood collected at baseline prior to the first randomization. However, the first set of SNP microarray results indicated a quality problem with approximately 50% of the DNA samples. A majority of the problematic samples were replaced with DNA extracted from redrawn blood. The remaining problematic samples were 'restored' using an Illumina FFPE Restoration procedure. A total of 3322 subjects (1638 from arm AA and 1684 from arm AB) were successfully genotyped. Among those, 1771 were from original blood samples, 1092 from redrawn blood samples and 459 from restored blood samples. The SUCCESS-A study is part of the Genomics and Randomized Trials Network (GARNET, http://www.garnetstudy.org) funded by the National Human Genome Research Institute (NHGRI). The overarching goal is to identify novel genetic factors that contribute to disease-free survival for breast cancer patients using different forms of chemotherapy through large-scale genome-wide association studies of treatment response in randomized clinical trials. The study was conducted at the University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nuremberg, Germany. Genotyping was performed at the Johns Hopkins University Center for Inherited Disease Research (CIDR). Data cleaning and harmonization were performed at the GARNET Coordinating Center at the University of Washington.

  Study phs000547

• California Pacific Medical Center Research Breast Health Cohort

  Description of Cohort: The California Pacific Medical Center (CPMC) Breast Health Cohort is a cohort study based at CPMC and is linked to the San Francisco Mammography Registry, one of the sites of the NCI-funded Breast Cancer Screening Consortium (U01CA063740). CPMC is a community hospital in San Francisco, which has one of the highest volumes for mammography in San Francisco. Between September 2004 and June 2007, >90,000 mammograms were performed at CPMC. The CPMC breast health cohort collects demographic and risk factor data on women receiving mammography through participation in the San Francisco Mammography Registry, as part of the Breast Cancer Screening Consortium (U01CA063740). The SFMR database collects information from all sources, including a questionnaire on demographic and risk factor information, the clinical results of the breast examination, the measures of breast density by Dr. John Shepherd and the women who agreed to donate a blood sample. By merging these various sources of information we have very efficiently developed a large sample of women who have donated blood and have had a measure of mamographic density. Blood Collection: Dr. Steve Cummings is leading an effort to collect and archive blood samples from women who are receiving mammography screening. All women who are sent for a screening mammogram at CPMC are considered eligible. Since the cohort began collecting blood samples in July 2004 until June 2007, samples have been collected from over 11,000 women. Measurement of Breast Density: Dr. John Shepherd is currently measuring breast density in a large fraction of the cohort using an automated approach with single X-ray absorptiometry. Dr. Shepherd has established a link with the CPMC mammography center that allows him to collect routine digital mammography information. Using the data from the mammogram, Dr. Shepherd and his group have developed the single X-ray absorptiometry (SXA) technique for measuring density which is described in more detail below. The table demonstrates the distribution of demographic variables and some breast cancer risk factors of women who donated blood and had a breast density measurement in the CPMC breast health cohort. Nearly 80% of the participants are Caucasian and most of the women are post-menopausal with a median age of ~52. Since it will be difficult to accrue a large enough sample from each ethnic group, our study will focus only on Caucasian women. Table: Demographic variables, reproductive history and family history of breast cancer among 2962 women participating in the CPMC cohort study who contributed blood samples between 1994-1997. Variable Median/Percentage Age (Median/IQR) 52 (46-59) Ethnicity Caucasian/White 0.76 Asian/Pacific Islander 0.141 Hispanic 0.029 Mixed Race/Ethnicity 0.039 African American/Black 0.022 American Indian 0.001 Other 0.009 First degree relative with breast cancer 0.17 Age at first birth Nulliparous 0.39 Age<20 0.043 Age>40 0.032 Age<30, ≥20 0.251 Age>30, ≤40 0.282 Measurement of Breast Density in Cohort: Measurement of breast density is accomplished using an automated technique for all mammograms obtained by Dr. Shepherd using Single X-Ray absorptiometry (SXA). SXA measurement of breast density is done on approximately 30% of all screening mammograms. Below we describe the method for measurement of breast density by SXA by Dr. Shepherd's group and its validation and association with breast cancer. As we demonstrate below, breast density, as measured by SXA, is an automated, highly reproducible measure of the density of breast tissue and is associated with breast cancer risk. SXA for Quantifying Breast Density: Single x-ray absorptiometry (SXA) was initially developed for measuring bone density. SXA can determine the fraction of each of two densities simultaneously using the fact the sample is a constant thickness, the thickness in known, and the total attenuation is known. In applying this technique to breast density, we assume a two compartment model: fat and non-fatty (fibroglandular tissue). We use a reference material composed of various concentrations of two materials: one which is the same density as fat and another which is the same density as fibroglandular tissue. The reference material (phantom) is placed in the X-ray field with each mammogram. We have been able to implement this in a way that is unintrusive to the patient and technologist at CPMC. Assuming this two-compartment model and a constant known breast thickness, we can then calculate the percent density at any region of the breast based on the assumption that % pixel grey-scale is proportion to the mass fractions of breast fat and lean tissue. If reference materials (a phantom) of fat and fibroglandular tissue are imaged with the patient's breast and the reference materials have the same thickness as the patient's breast, then the breast's grey-scale values can be converted to fat/fibroglandular mass fractions by interpolating between those two references. The total percent density is found by averaging the volume fraction over all breast pixels. The phantom being used for breast density assessment at CPMC began to be used in September 2004. The phantom does not have to be manipulated by the technologist and stays attached on the mammography device during standard craniocaudal (CC) views. Thus it creates minimal to no interference with the clinical mammogram. Reproducibility of breast density measures: Traditional measures of mammographic density require some human interpretation. A human reader outlines the area perceived to be dense and a computer then calculates the percent area outlined as a percent of the entire image. Thus, while traditional mammographic density is associated with breast cancer risk, it has some limitations. In a study by Drs. Shepherd, Kerlikowske, et al., the correlation coefficient (Pearson's R) between different readers was 0.8-0.9. In contrast to the traditional mammographic density measurement, the SXA measurement is fully automated and, therefore, the reproducibility of the measurement is higher. Dr. Shepherd and colleagues have performed a replication study of SXA as a measurement of breast density. They have estimated the correlation coefficient of the SXA measurement of breast density to be >0.98. Thus, as expected for an automated measure, SXA is a highly reproducible measure of mammographic breast density. Drs. Shepherd and Kerlikowske have recently analyzed the association between breast cancer risk and breast density as measured by SXA (Shepherd et al., Cancer Epi Biomarkers and Prev, 2011, PMID: 21610220). They found that women in the highest quintile of % volumetric density had an odds ratio of 4.1 (95% CI: 2.3 - 7.2) for breast cancer risk compared to women in the lowest quintile of volumetric density. Thus volumetric density appears to be a highly reproducible, automated measure of breast cancer.

  Study phs000395

• Mucociliary Clearance Consortium (MCC) Longitudinal Study of Primary Ciliary Dyskinesia: Participants 5-18 Years of Age

  The purpose of this longitudinal study protocol is to define age-related prevalence of phenotypic characteristics and the progression of key features of lung disease in participants with one of these disorders, Primary Ciliary Dyskinesia (PCD). In PCD, the abnormal structure and function of cilia results in impaired clearance of secretions and consequent obstruction and chronic recurrent infection in the airways, sinuses and middle ears. Although it seems likely that the onset of PCD airway disease occurs early in childhood, as has been reported for cystic fibrosis (CF), the clinical course of PCD lung disease is not well defined, nor, is the time course of emergence of specific microbial pathogens, or the age of onset and rate of progression of airway disease and bronchiectasis. This longitudinal study is designed to define the rate of progression of PCD lung function in participants between 5-18 years of age using spirometry which tracks well with lung impairment and prognosis in other disorders of the airways such as cystic fibrosis. This longitudinal protocol will also systematically track other specific outcomes, including pathogens infecting the airways (assessed by respiratory cultures), and age at onset and progression of airway damage and bronchiectasis (assessed by high-resolution computerized tomography, HRCT, of the chest). Blood will be collected for DNA to test for genetic mutations. The blood samples will be processed to establish a cell line (lymphocyte transformation) for a source of DNA for future genetic studies, in participants with disease. For participants unable to provide a blood sample, a buccal sample will be obtained for DNA.

  Study phs000596

• GEnetics of Nephropathy - an International Effort (GENIE) GWAS of Diabetic Nephropathy in the UK GoKinD and All-Ireland Cohorts

  Diabetic kidney disease, or diabetic nephropathy (DN), is one of the leading causes of end-stage renal disease in the United States and worldwide. DN is a common complication of long-standing type 1 and type 2 diabetes. The clinical course is characterized by development of proteinuria and gradual loss of kidney function. Although existing treatments that decrease proteinuria have been shown to moderately abate progression of diabetic kidney disease, many affected patients, who do not die from cardiovascular disease, go on to develop terminal renal failure, necessitating costly renal replacement therapies, such as dialysis and renal transplantation. Type 1 diabetes (T1D) can have its onset in childhood and affected individuals often develop end-stage renal disease in early adulthood, leading to further loss of quality of life. The genetic basis of the disease is not well understood. The GENIE (GEnetics of Nephropathy an International Effort) consortium was initiated to perform the most comprehensive and well powered DN susceptibility genome wide association study (GWAS) analysis to date, using the largest collection of type 1 diabetics with and without kidney disease across four study cohorts. The UK-ROI samples were genotyped as part of this project. UK-ROI Sample Description The UK-ROI collection consists of samples derived from the Republic of Ireland (Dr. Catherine Godson, PI, at University College, Dublin, Ireland) and the United Kingdom (Warren 3 and Genetics of Kidneys in Diabetes UK, UK GoKinD, Dr. Alexander P. Maxwell, PI, at Queen's University of Belfast, UK). All study subjects met the inclusion criteria: white individuals with T1D, diagnosed before 31 years of age, whose parents and grandparents were born in the British Isles.

  Study phs000389

• The EVE Asthma Genetics Consortium: Building Upon GWAS

  The EVE Asthma Genetics Consortium comprises U.S. investigators who have conducted genome-wide association studies (GWAS) of asthma; the main objective is to combine results of individual studies to increase the overall power to identify loci for asthma and asthma-associated traits. The consortium includes investigators at 9 U.S. institutions with GWAS results for >10,000 individuals representing European American, African American, African Caribbean, U.S. Hispanic, and Mexican populations and includes the following studies: The Study of Asthma Phenotypes and Pharmacogenomic Interactions by Race-Ethnicity (SAPPHIRE) The Genetic Research on Asthma in the African Diaspora (GRAAD) Study The Genetics of Asthma in Latino Americans (GALA 1) Study The Childhood Asthma Management Program (CAMP) The Childhood Asthma Research and Education (CARE) Network The Children's Health Study (CHS) Mexico City Childhood Asthma Study (MCCAS) The Chicago Asthma Genetics Study (CAG)* The National Heart, Lung, and Blood Institute Collaborative Studies of the Genetics of Asthma (CSGA)* The Severe Asthma Research Program (SARP)* (*CAG, CSGA, and SARP are part of the NHLBI-supported SNP Typing for Association with Multiple Phenotypes from Existing Epidemiologic Data (STAMPEED) consortium.) For the GWAS, we developed a common set of >1 million genotyped and imputed SNPs from the EVE Asthma Genetics Consortium to be tested for association with asthma and associated phenotypes in all samples and combined p-values for a grand meta-analysis for asthma gene discovery. A subset of 296 individuals representing African American, European American, and Latino ancestry were selected for whole genome sequencing. The broad goals of this project were to characterize the genetic architecture of asthma and associated quantitative phenotypes (e.g., lung function, total serum IgE) in ethnically diverse populations from the U.S., Mexico, Puerto Rico, and Barbados.

  Study phs001156

• Characterization of Human Transcriptome by Computational and HTS Approaches

  We have deeply sequenced total RNA and whole exome of six different post-mortem human snap-frozen tissues (brain, liver, lung, striated muscle, kidney, heart) from three unrelated healthy Caucasian individuals (males, aged 47-54) with the aim to characterize post-transcriptional events due to alternative splicing and RNA editing. In addition, to facilitate the detection of RNA editing sites we have also resequenced the entire genome of each individual. Data are also used to investigate tissue-specific abundance of mitochondrial DNA from exomes and its correlation with mitochondrial transcription, mass and respiratory activity. Tissues were obtained from Cureline (South San Francisco, CA, USA). DNA and RNA were purified using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) and the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), respectively. Exome capture was performed using the TruSeq Exome Enrichment Kit (Illumina, San Diego, CA) and the sequencing was carried out at IGA Technology Services in Udine (Italy) on Illumina HiSeq 2000 (generating for each tissue approximately 40 millions of 100bp paired-end reads). Strand-oriented RNA reads (2x100bp) were generated using the TruSeq Stranded Total RNA Sample Prep Kit (Illumina, San Diego, CA, USA) and sequenced at IGA Technology Services in Udine (Italy) on Illumina HiSeq 2000 platform. Whole genome resequencing was performed at Personal Genomics in Italy on an Illumina HiSeq 2000 platform (2x100bp) with a mean coverage of 30X. MiRNA-Seq was instead performed at IBBE Institute (Italy) using the TruSeq Small RNA Sample Prep Kit on Illumina MiSeq platform. Preliminary bioinformatics analyses have been performed to check quality using FASTQC software and inconsistent read regions have been trimmed using trim_galore. All reads were mapped onto the human reference genome (version hg19) using GSNAP.

  Study phs000870

• Heart and Vascular Health Study (HVH)

  Objectives The Heart and Vascular Health Study (HVH) is a case-control study of risk factors for the development of myocardial infarction (MI), stroke, venous thrombosis (VT), and atrial fibrillation (AF). The study setting is Group Health, an integrated health care delivery system in Washington State. Background The HVH originated in 1988 with the examination of risk factors for MI. Over the ensuing years, the study has been funded by a series of grants, which have added case subjects with stroke, VT, and AF, and used a common control group. Study aims have focused on the associations of medication use with cardiovascular events. Starting in 1997, the study aims expanded to include genetic associations with cardiovascular disease. Participants recruited in 2009 or later and who provided blood samples for genetic analysis were asked for consent to deposit genetic and phenotype data in dbGaP. Design As part of the HVH study, case subjects were identified by searching for ICD-9 codes consistent with MI, stroke, VT, or AF, and medical records were reviewed to confirm the diagnosis. Control subjects were identified at random from the Group Health enrollment and were matched to MI cases. All subjects have an index date. For cases, the index date was assigned as the date that the cardiovascular event (MI, stroke, VT, or AF) came to clinical attention. For controls, the index date was a random date within the range of the case index dates. For both cases and controls, information was collected from the inpatient and outpatient medical record, by telephone interview with consenting survivors, and from the Group Health pharmacy and laboratory databases. Consenting participants provided a blood specimen. Genetic Research Genetic factors underlying cardiovascular disease are studied using DNA isolated from the blood samples.

  Study phs001013

• Diabetes Control and Complications Trial (DCCT) and Epidemiology of Diabetes Interventions and Complications Study (EDIC)

  The Diabetes Control and Complications Trial (DCCT, 1982-93) and the Epidemiology of Diabetes Interventions and Complications follow-up study (EDIC, 1994-2016) have been ongoing for more than twenty years. After a mean follow-up of approximately 16 years, the DCCT-EDIC cohort of 1,441 Type 1 diabetics had remained remarkably complete with more than 90% of the original cohort being actively followed. Taken together, the DCCT clinical trial and subsequent EDIC longitudinal follow-up provide a uniquely rich source of information on the impact of intensive therapy on glycemia and the its long-term complications for persons with Type 1 diabetes. The DCCT was a multicenter, randomized clinical trial (1, 2) designed to compare intensive with conventional diabetes therapy with regard to their effects on the development and progression of the early complications of Type 1 diabetes. The DCCT trial found that "intensive therapy effectively delays the onset and slows the progression of diabetic retinopathy, nephropathy, and neuropathy" in patients with Type 1 diabetes (1). The goal of the EDIC follow-up was to examine the longer term effects of the original DCCT interventions, especially as they apply to late-occurring complications, such as cardiovascular disease and more advanced stages of retinal and renal disease (3). The EDIC study has been remarkably fruitful in discovering the long term "imprinting" effects (metabolic memory) of the previous intensive therapy, and in delineating the interactions among risk factors with regard to microvascular complications (4-6). In addition, EDIC established, for the first time, the role of intensive therapy and chronic glycemia on atherosclerosis (7-9). Note: This study description has been prepared using materials authored by the DCCT-EDIC Data Coordinating Center. DETAILED DESCRIPTION OF STUDY

  Study phs000086

• Human Gene Expression Patterns Associated with Experimental P. falciparum Infection

  Description: Experimental exposure to malaria parasites can lead to development of protective immunity, providing a foothold for the development of a malaria vaccine. The goal of this study is to investigate immune transcriptional profiles associated with malaria protective immune responses induced by experimental Chemo-Prophylaxis and Sporozoites (CPS) immunization of P. falciparum-naive human volunteers. Samples for this study were obtained from a CPS-immunization study conducted in 2011 (ClinicalTrials.gov Identifier: NCT01218893), where healthy volunteers received CPS-immunization with bites from different numbers of P. falciparum-infected mosquitoes (three times 15 (n=5), 10 (n=9) or 5 (n=10)). Five control subjects received uninfected mosquito bites under chloroquine prophylaxis. Fifteen weeks after discontinuation of chloroquine prophylaxis, all volunteers were challenged by exposure to infected mosquito bites. Study Design: A maximum of 30 volunteers were divided into four groups. All volunteers received weekly chloroquine prophylaxis for a period of 13 weeks (91 days). During these 13 weeks, on days 8, 36 and 64 they were exposed to the bites of 15 mosquitoes. Group 1 (n=5, positive control) received three CPS immunizations by 15 mosquitoes infected with P. falciparum sporozoites. Group 2 (n=10) received three times 10 bites from infected mosquitoes and 5 bites from uninfected mosquitoes. Group 3 (n=10) received three times 5 bites from infected mosquitoes and 10 bites from uninfected mosquitoes. Group 4 (n=5), the negative control, received three placebo immunizations with 15 bites of uninfected mosquitoes. Fifteen weeks after discontinuation of chloroquine prophylaxis, all 30 volunteers were challenged at day 196 by the bites of 5 infectious mosquitoes and followed for 21 days. All subjects were treated with chloroquine 21 days after challenge or whenever they had a positive thick smear during that period.

  Study phs001346

• Columbia University Study of Caribbean Hispanics with Familial and Sporadic Late Onset Alzheimer's disease

  Compared to Caucasians residing in the same community the incidence rate of Alzheimer's disease is approximately twice as high in Caribbean Hispanics. Moreover, Caribbean Hispanics represent a homogenous population with only a few founders. Replication of genetic associations in other ethnic groups provides supporting evidence that a putative gene is involved in the disease pathogenesis, and when different allelic variants within the same genes are associated with disease it can help to localize the pathogenic variant. SORL1 is an example of a candidate gene that was first identified in Hispanics and then confirmed in multiple ethnic and racial groups in a meta-analysis. For this project, we are genotyping 704 individuals in families multiply affected by Alzheimer's disease (166 families). These families were recruited in the United States, Puerto Rico and the Dominican Republic. In addition, we are genotyping 2491 individuals (960 patients with sporadic Alzheimer's disease and 1531 unrelated controls) phenotyped in a similar fashion totaling 3195 individuals. Both cohorts are followed at regular intervals of 18 to 24 months, and potential phenotypes available other than those related to Alzheimer's disease include body mass index, measured blood pressure, neurological history and examinations. More importantly, both studies are funded through 2014 and additional phenotypes could be added in successive waves. We propose a genome wide association (GWA) study of Alzheimer's disease and longitudinal changes in cognition and other age-related neurological and medical phenotypes. The goal will be to identify the chromosomal locations of genes underlying this disease and its related endophenotypes.

  Study phs000496

• Genomic Studies in Charcot-Marie-Tooth Disease

  The various forms of Charcot-Marie-Tooth disease (CMT) comprise a genetically heterogeneous set of peripheral neuropathies. Currently, >90 different CMT genes have been identified; yet, for the axonal CMT2 subtype these genes explain only 30-40% of the genetic effect. In particular, the application of exome sequencing has led to an unprecedented pace in identifying about half of those genes in the past five years. We have been at the forefront of this development with 22+ published new gene identifications in the past seven years. This success was only possible through broad national and international collaborations, data sharing, and the development of advanced analysis tools and methods. We also learned that despite an accumulation of exome sequencing data, over 50% of axonal CMT patients remain undiagnosed, suggesting even more genes are involved; additionally, our preliminary data also support the existence of non-coding variations as Mendelian allele contributors. In this study, we will continue our highly impactful work in CMT family recruitment, exome sequencing and traditional gene identification. In addition, we will expand the genetic studies to whole genome sequencing with a focus on families already explored unsuccessfully in whole exome studies. We further plan to perform the first large rare variant burden analysis in the rare disorder CMT2. This is only possible through the exceptional clinical resources we have built over the past decade. Finally, with many CMT genes available, we will perform statistical network analyses on a CMT-ome to identify gene modules and pathways that will be the starting point of polypharmacology, and provide insight into pathophysiology of peripheral nerve degeneration. All data will be available in real-time to an existing network of CMT investigators worldwide, and deposited in dbGaP annually.

  Study phs003389

• Hispanic Community Health Study /Study of Latinos (HCHS/SOL)

  The Hispanic Community Health Study / Study of Latinos (HCHS/SOL) is a multi-center epidemiologic study in Hispanic/Latino populations to determine the role of acculturation in the prevalence and development of disease, and to identify risk factors playing a protective or harmful role in Hispanics/Latinos. The study is sponsored by the National Heart, Lung, and Blood Institute (NHLBI) and six other institutes, centers, and offices of the National Institutes of Health (NIH). The goals of the HCHS/SOL include studying the prevalence and development of disease in Hispanics/Latinos, including the role of acculturation, and identifying disease risk factors that play protective or harmful roles in Hispanics/Latinos. A total of 16,415 persons of Cuban, Dominican, Mexican, Puerto Rican, Central American, and South American backgrounds were recruited through four Field Centers affiliated with San Diego State University, Northwestern University in Chicago, Albert Einstein College of Medicine in the Bronx area of New York, and the University of Miami. Seven additional academic centers serve as scientific and logistical support centers. Study participants aged 18-74 years took part in an extensive clinic exam and assessments to ascertain socio-demographic, cultural, environmental and biomedical characteristics. Annual follow-up interviews are conducted to determine a range of health outcomes. The HCHS SOL Cohort is utilized in the following dbGaP sub-studies. To view genotypes, other molecular data, and derived variables collected in these sub-studies, please click on the following sub-studies below or in the "Sub-studies" section of this top-level study page phs000810 HCHS SOL Cohort. phs000555 PAGE CALiCo SOL phs000880 HCHS/SOL Omics in Latinos (Ola)

  Study phs000810

• ChiLDReN/BA: Genetic Studies of Biliary Atresia in the Childhood Liver Disease Research Network

  Biliary atresia (BA) is a progressive necroinflammatory process initially involving the extra-hepatic biliary tree. Little is known about the factors that cause BA or the factors that influence disease progression. A variety of genetic, autoimmune, and environmental influences have been hypothesized to be important. Most studies to date have focused on the neonate and young child with BA, yet the older surviving child with BA can provide important information about genetics as well as natural history.The Childhood Liver Disease Research Network (ChiLDReN) conducts two longitudinal and observational studies that include BA patients: Biliary Atresia Study in Infants and Children (BASIC) and A Prospective Database of Infants with Cholestasis (PROBE). Using samples from participants in these two protocols, we have formed the largest known data set of biliary atresia patients in the world. ChiLDReN is performing a series of genomic analyses, with subsequent analyses being released as subsequent versions of this registration, phs003356. Version 1 concerns PKD1L1.About the PKD1L1 Analysis (phs003356.v1): While multiple factors have been implicated in the perinatal biliary injuries that characterize BA, a definitive etiology has not yet been established. This study is part of a larger project to characterize the largest cohort of pediatric BA patients through whole-exome sequencing (WES). In this analysis, we examine the WES of those with various laterality defects to determine whether genetic factors could be identified to explicate the etiopathogenesis of BA in biliary atresia splenic malformation (BASM) syndrome. We specifically explored variations in the PKD1L1 gene, which recently has been identified in mouse models as playing a role in laterality.

  Study phs003356

• Somatic Mutation in Normal Bladder Study

  Adult human somatic tissues are landscapes of mutant clones, but little is known about how these landscapes are shaped by known cancer risk factors. The goal of this study was to determine how the main bladder cancer risk factors (sex and smoking) change the mutational landscape of the normal urothelium. We collected normal urothelium from 45 donors at autopsy by brushing ~2cm² of the surface epithelium in the upper part (dome) and lower part (trigone) of the bladder (79 samples total). To identify mutations with high sensitivity we employed ultradeep (~5000x) DNA error-correcting duplex-sequencing on a panel of 15 protein coding genes and the TERT promoter, previously described to be under positive selection in normal urothelium or relevant to bladder cancer. We identified thousands of clonal driver mutations, which demonstrate pervasive positive selection in the normal urothelium. Males showed a significantly higher number of driver truncating mutations in RBM10, CDKN1A and ARID1A, despite similar rates of non-coding mutations, indicating stronger positive selection and clonal expansion in the male urothelium. We also discovered clonal expansions driven by activating TERT promoter mutations in the normal bladder, which were strongly associated with age and smoking. These results demonstrate that sex and smoking shape the clonal landscape of the normal bladder and indicate that the high mutational content of normal tissues might be leveraged to study the effect of mutations in vivo (i.e. natural saturation mutagenesis). We will deposit in dbGaP de-identified relevant clinical information from the 45 donors and the fastq files derived from duplex sequencing of the 79 samples in the study. We will also deposit fastq files derived from duplex sequencing of 3 control cord blood samples.

  Study phs004105

• CPTAC Proteogenomic Study

  Recently, significant progress has been made in characterizing and sequencing the genomic alterations in statistically robust numbers of samples from several types of cancer. For example, The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC) and other similar efforts are identifying genomic alterations associated with specific cancers (e.g., copy number aberrations, rearrangements, point mutations, epigenomic changes, etc.) The availability of these multi-dimensional data to the scientific community sets the stage for the development of new molecularly targeted cancer interventions. Understanding the comprehensive functional changes in cancer proteomes arising from genomic alterations and other factors is the next logical step in the development of high-value candidate protein biomarkers. Hence, proteomics can greatly advance the understanding of molecular mechanisms of disease pathology via the analysis of changes in protein expression, their modifications and variations, as well as protein=protein interaction, signaling pathways and networks responsible for cellular functions such as apoptosis and oncogenesis. Realizing this great potential, the NCI launched the third phase of the CPTC initiative in September 2016. As the Clinical Proteomic Tumor Analysis Consortium, CPTAC continues to define cancer proteomes on genomically-characterized biospecimens. The purpose of this integrative approach was to provide the broad scientific community with knowledge that links genotype to proteotype and ultimately phenotype. In this third phase of CPTAC, the program aims to expand on CPTAC II and genomically and proteomically characterize over 2000 samples from 10 cancer types (Lung Adenocarcinoma, Pancreatic Ductal Adenocarcinoma, Glioblastoma Multiforme, Acute Myeloid Leukemia, Clear cell renal Carcinoma, Head and Neck Squamous Cell Carcinoma, Cutaneous Melanoma, Sarcoma, Lung Squamous Cell Carcinoma, Uterine Corpus Endometrial Carcinoma) .Germline DNA is obtained from blood and Normal control samples for proteomics varied by organ site. All cancer samples were derived from primary and untreated tumor.

  Study phs001287

• CCR8-targeted specific depletion of clonally expanded Treg cells in tumor tissues evokes potent tumor immunity with long-lasting memory

  Foxp3-expressing CD25+CD4+ regulatory T cells (Tregs) are abundantly infiltrating into tumor tissues, hindering effective anti-tumor immunity. Here, assuming that such tumor Tregs were clonally expanding by recognizing tumor-associated antigens, we attempted to characterize them by their gene expression profiling and T-cell receptor (TCR) clonotyping at the single-cell level. We found that they predominantly expressed the chemokine receptor CCR8: ~40% of tumor Tregs, less than 10% of Tregs in other tissues, and few tumor-infiltrating conventional T cells expressed CCR8 in mice and humans. CCR8+ tumor Tregs were phenotypically highly differentiated and functionally stable. One injection of anti-CCR8 mAb with high antibody-dependent cellular cytotoxic activity was indeed able to specifically deplete clonally expanded tumor Tregs and thereby eradicate established tumors in mice. The depletion predominantly expanded tumor-infiltrating CD8+ effector T cells, which were different in activated or exhausted patterns from those seen after immune checkpoint blockade by anti-PD-1 mAb. The treatment also evoked potent secondary immune responses against the same tumor cell line inoculated several months after tumor eradication, indicating that the anti-CCR8-induced memory-type tumor-reactive effector T cells were, once induced, resistant to recovered Treg suppression. In contrast to such evocation of potent tumor immunity, anti-CCR8 treatment scarcely elicited discernable autoimmunity in mice, contrasting with a similar treatment for systemic whole Treg depletion, which induced histologically and serologically evident autoimmunity. Thus, temporary and specific depletion of clonally expanded Tregs in tumor tissues by cell-depleting anti-CCR8 mAb is sufficient to evoke potent tumor immunity with long-lasting memory and without serious autoimmunity.

  Study JGAS000312

• Genome and gene analysis of gastrointestinal cancer and elucidation of its clinicopathological significance

  Cancer develops through the accumulation of genetic and epigenetic aberrations. To identify sequential molecular alterations that occur during the development of hepatocellular carcinoma (HCC), we molecularly compared 52 early and 108 overt HCC samples by genome sequencing. Gene mutations in the p53/RB1 pathway, WNT pathway, MLLs, SWI/SNF complexes, and AKT/PI3K pathway were common in HCC. In the early phase of all entities, TERT was the most frequently upregulated gene owing to diverse mechanisms. Despite frequent somatic mutations in driver genes, including CTNNB1 and TP53, early HCC was a separate molecular entity from overt HCC as each had a distinct expression profile. Notably, WNT target genes were not activated in early HCC, regardless of CTNNB1 mutation status, because ?-catenin did not translocate into the nucleus due to the E-Cadherin/?-catenin complex at the membrane. Conversely, WNT targets were definitively upregulated in overt HCC with CTNNB1 mutation associated with downregulation of CDH1 and hypomethylation of CpG islands in the target genes. Similarly, cell cycle genes downstream of the p53/RB pathway were upregulated only in overt HCC with TP53 and/or RB1 gene mutations associated with chromosomal deletion of 4q and/or 16q. Differing from the expression profile, HCC was epigenetically distinguished into four subclasses: normal-like methylation, global-hypomethylation (favorable prognosis), stem-like methylation (poor prognosis), and CpG island methylation. These methylation statuses were globally maintained through HCC progression. Taken together, additional molecular events besides driver gene mutations cooperatively contribute to transcriptional activation of downstream targets as HCC progresses, according to the methylation status of the associated genes.

  Study JGAS000233

• How upcycled prostate cancer sequences enabled key findings on telomeres length

  The length of telomeres, the chromosomes’ end tails, as depicted in the figure below, physiologically shorten with aging. This means that younger cells have longer telomeres compared to their aged counterparts; one could say that the length of cells’ telomeres is a measure of their replicative power. Cancer cells are known to be able to turn this into their favour, and expand their replicative immortality through telomere lengthening and maintenance. Despite being a well-studied pan-cancer hallmark, the relation of the telomeres length to specific cancer genomic features is poorly understood. Julia Livingstone and her colleagues from P.C. Boutros' team in Los Angeles focused their work on prostate cancer. To investigate the relations between telomere length and the clinico-genomics of prostate tumours, they used whole genome sequencing (WGS) of 392 published tumour–reference pairs previously deposited at the EGA under accession numbers EGAS00001000400 and EGAS00001000900. Their work was published on Nature Communications in November 2021. They determined the telomere length of the sequencing sample, from both tumour and non-tumour adjacent tissues, and subsequently ran statistical analysis to investigate possible correlation between the telomeres length with several cancer features. The team discovered that telomeres length does not correlate with any specific driver gene mutation, but it does with the overall frequency of single nucleotides variants in a sample. This finding suggests that tumours with shorter telomeres accumulate more mutations without strong selective pressures for specific ones. While proliferation rate did not correlate directly with telomere length, methylation of almost half of all genes strongly did. The team’s results are massive and reveal a complicated and fascinating relation of different clinical and genomic features of prostate cancer with telomere length. In the paper discussion, they smartly consider different possibilities. As noted above, all these amazing results have been obtained analysing previously sequenced samples. Remarkably, the researchers who generated the samples did not focus at all on telomeres length. They indeed studied specific genes mutations and the features of early and late-onset prostate cancer. This proves, once again, the power of sequencing data re-use. This data is tremendously rich in information, and can hardly be exhausted with any number of analyses. Generating sequencing data, especially Whole Genome Sequencing, even better if tumor-reference paired, is like shopping every last article at the grocery store. You can then cook several recipes, or prepare a seven-course dinner, and still have ingredients left for someone else to borrow and cook something you have not thought about, a recipe that is just not in your cookbook. Noteworthy, the data retrieved from the EGA for this paper is associated with rich and complete metadata, meaning pheno-clinical information regarding the sample donors. As Julia Livingstone and her team wrote, using rich clinical annotations enabled them to assess the relationship between telomere length and patients outcome. This highlights once again the importance of reporting and annotating metadata correctly; It might feel like extra work for the researchers depositing their data, but it really has a huge value. Our team at the EGA is currently working to improve the metadata annotation process, with the aim to maximize the benefits for the scientific community.

  Blog prostate-cancer-sequences-enabled-key-findings-on-telomeres-length

• Discovery and validation of an ancillary genomic test of malignancy for primary melanocytic tumors

  Background:Pathological diagnosis of melanocytic tumors can be difficult and prone to error. More accurate and reliable ancillary investigations than those currently available are required to improve diagnostic accuracy. Goals: (1) to generate a genomic model for distinguishing benign (nevi) from malignant (melanoma) melanocytic skin tumors, developed from a customized gene panel evaluated on a cohort of 250 nevi and melanomas. (2) To evaluate the performance of the genomic model in melanocytic tumors of uncertain diagnosis/behavior. Details: The cohort of 250 melanomas and naevi was split into a discovery set and a validation set. Our genomic model obtained an area under the curve of 95% in the discovery cohort and 96% for the validation cohort. Based on a Youden index threshold, the resulting classifier shows high specificity (>95% in the discovery and validation cohorts), with sensitivities >80% in the discovery and validation cohorts. Evaluation of the genomic model on a cohort of 110 melanocytic tumors identifies key driver mutations in agreement with the likely pathway of origin of the tumors. The probability of melanoma (p=0.02) from the genomic model was significantly associated with pathology-based estimates of malignancy for melanocytic tumors lacking pathway-defining genomic aberrations (conventional tumors). The genomic model identified 8/36 (22%) conventional tumors with unresolved malignancy to have a high probability of melanoma, with 7/8 of these cases showing negative/non-aberrant results with either fluorescent in-situ hybridization, PRAME and/or p16 immunostains. Furthermore, no evidence of malignant behavior based on the genomic model was seen in 4 conventional borderline tumors with more than 1 year follow-up. Conclusion: This genomic model can become a clinically useful ancillary tool that is highly specific for differentiating melanomas from nevi.

  Study EGAS50000000887

• METABRIC

  Solid tumors are complex tissues composed of a mixture of cancer and normal cells, which complicates the interpretation of their molecular profiles. Normal cell contamination can dilute cancer cell information and tissue architecture is generally not reflected in molecular assays. To address these challenges, we developed a computational approach based on standard Haematoxylin and Eosin-stained sections and demonstrated its power in a discovery cohort of 323 breast tumors and an independent validation cohort of 241 tumors. First, to deconvolute cellular heterogeneity and detect subtle genomic aberrations, we introduced an algorithm based on tumor cellularity to increase the comparability of copy-number profiles between samples. Second, we demonstrated that a predictor for survival integrating image-based and molecular features significantly outperforms classifiers based on single data types. Third, we described and validated a novel, independent prognostic factor based on quantitative analysis of spatial patterns between stromal cells, which are not detectable by molecular assays. Our quantitative methods refine and complement molecular assays of tumor samples and could benefit all large-scale cancer studies.First, to deconvolute cellular heterogeneity and detect subtle genomic aberrations, we introduced an algorithm based on tumor cellularity to increase the comparability of copy-number profiles between samples. Second, we demonstrated that a predictor for survival integrating image-based and molecular features significantly outperforms classifiers based on single data types. Third, we described and validated a novel, independent prognostic factor based on quantitative analysis of spatial patterns between stromal cells, which are not detectable by molecular assays. Our quantitative methods refine and complement molecular assays of tumor samples and could benefit all large-scale cancer studies.

  Study EGAS00000000098

• Enhanced detection of circulating tumor DNA by fragment size analysis

  Existing methods to improve detection of circulating tumor DNA (ctDNA) have focused on sensitivity for detecting genomic alterations but have rarely considered the biological properties of plasma cell-free DNA (cfDNA). We hypothesized that differences in fragment lengths of circulating DNA could be exploited to enhance sensitivity for detecting the presence of ctDNA and for non-invasive genomic analysis of cancer. We surveyed ctDNA fragment sizes in 344 plasma samples from 200 cancer patients using low-pass whole-genome sequencing (0.4×). To establish the size distribution of mutant ctDNA, tumor-guided personalized deep sequencing was performed in 19 patients. We detected enrichment of ctDNA in fragment sizes between 90–150 bp, and developed methods for in vitro and in silico size selection of these fragments. Selecting fragments between 90–150 bp improved detection of tumor DNA, with more than 2-fold median enrichment in >95% of cases, and more than 4-fold enrichment in > 10% of cases. Analysis of size-selected cfDNA identified clinically actionable mutations and copy number alterations that were otherwise not detected. Identification of plasma samples from patients with advanced cancer was improved by predictive models integrating fragment length and copy number analysis of cfDNA, with AUC> 0.99 compared to AUC < 0.80 without fragmentation features. Increased identification of cfDNA from patients with glioma, renal, and pancreatic cancer was achieved with AUC > 0.91, compared to AUC < 0.5 without fragmentation features. Fragment size analysis and selective sequencing of specific fragment sizes can boost ctDNA detection. This could reduce the required depth of cfDNA sequencing for clinical applications, earlier diagnosis and study of tumor biology.

  Study EGAS00001003258

• The light chain IgLV3-21 defines a new poor prognostic subgroup in Chronic Lymphocytic Leukemia: results from a multicenter study

  Purpose: Unmutated (UM) immunoglobulin heavy chain region (IgHV) status or IgHV3-21 gene usage is associated with poor prognosis in chronic lymphocytic leukemia (CLL). Interestingly, IgHV3-21 is often co-expressed with light chain IgLV3-21 which is potentially able to trigger cell-autonomous BCR-mediated signaling. However this light chain has never been characterized independently of the heavy chain IgHV3-21.Experimental Design: we performed total RNA sequencing to characterize IgLV3-21 patients (n=32) and investigated IgLV3-21 prognostic impact in terms of treatment-free (TFS) and overall (OS) survival in 3 other independent cohorts for a total of 813 patients where IgLV3-21 presence was tested by real-time PCR and confirmed by Sanger sequencing.Results: Using total RNA sequencing to characterize 32 patients with high-risk CLL, we found a high frequency (28%) of IgLV3-21 rearrangements. Gene set enrichment analysis revealed that these patients present higher expression of genes responsible for ribosome biogenesis and translation initiation (P<0.0001) and MYC target genes (P=0.0003). Patients with IgLV3-21 rearrangements displayed a significantly shorter TFS and OS (P<0.05), in particular in IgHV mutated patients. In each of the 3 independent validation cohorts (n=813), we showed that IgLV3-21 rearrangements -similar to UM IgHV status- conferred poor prognosis compared to mutated IgHV (P<0.0001). Importantly, we confirmed in multivariate analysis that this was independent of IgHV mutational status or subset #2 stereotyped receptor (P<0.0001). Conclusions: We therefore demonstrate for the first time that a light chain can impact on CLL prognosis and that IgLV3-21 light chain usage defines a new poor prognostic subgroup in CLL.

  Study EGAS00001002894

• A genome scan for genes underlying adult body size differences between Central African hunter-gatherers and farmers.

  The evolutionary and biological bases of the Central African pygmy phenotype, a characteristic of rainforest huntergatherers defined by reduced body size compared with neighboring farmers, remain largely unknown. Here, we perform a joint investigation in Central African hunter-gatherers and farmers of adult standing height, sitting height, leg length, and body mass index (BMI), considering 358 hunter-gatherers and 169 farmers with genotypes for 153,798 SNPs. In addition to reduced standing heights, hunter-gatherers have shorter sitting heights and leg lengths and higher sitting/standing height ratios than farmers and lower BMI for males. Standing height, sitting height, and leg length are strongly correlated with inferred levels of farmer genetic ancestry, whereas BMI is only weakly correlated, perhaps reflecting greater contributions of non-genetic factors to body weight than to height. Single- and multi-marker association tests identify one region and eight genes associated with hunter-gatherer/farmer status, and 24 genes associated with the height-related traits. Many of these genes have putative functions consistent with roles in determining their associated traits and the pygmy phenotype, and they include three associated with standing height in non-Africans (PRKG1, DSCAM, MAGI2). We find evidence that European height-associated SNPs or variants in linkage disequilibrium with them contribute to standing- and sitting-height determination in Central Africans, but not to the differential status of hunter-gatherers and farmers. These findings provide new insights into the biological basis of the pygmy phenotype, and they highlight the potential of cross-population studies for exploring the genetic basis of phenotypes that vary naturally across populations. Pemberton et al. Human Genetics 2018 https://doi.org/10.1007/s00439-018-1902-3

  Study EGAS00001002975

• Combined MEK and BCL-2/XL inhibition is effective in high-grade serous ovarian cancer patient-derived xenograft models and BIM levels are predictive of responsiveness

  Most patients with late stage high-grade serous ovarian cancer (HGSOC) initially respond to chemotherapy but inevitably relapse and develop resistance, highlighting the need for novel therapies to improve patient outcomes. The MEK/ERK pathway is activated in a large subset of HGSOC, thus making it an attractive therapeutic target. Here, we systematically evaluated the extent of MEK/ERK pathway activation and efficacy of pathway inhibition in a large panel of well-annotated HGSOC patient-derived xenograft (PDX) models. The vast majority of models were nonresponsive to the MEK inhibitor cobimetinib (GDC-0973) despite effective pathway inhibition. Proteomic analyses of adaptive responses to GDC-0973 revealed that GDC-0973 upregulated the pro-apoptotic protein BIM, thus priming the cells for apoptosis regulated by BCL2-family proteins. Indeed, combination of both MEK inhibitor and dual BCL-2/XL inhibitor (ABT-263) significantly reduced cell number, increased cell death and displayed synergy in vitro in most models. In vivo, the GDC-0973 and ABT-263 combination was well tolerated and resulted in greater tumor growth inhibition than single agents. Detailed proteomic and correlation analyses identified two subsets of responsive models – those with high BIM at baseline that was increased with MEK inhibition and those with low basal Bim and high pERK levels. Models with low BIM and low pERK were non-responsive. Our findings demonstrate that combined MEK and BCL-2/XL inhibition has therapeutic activity in HGSOC models and provide a mechanistic rationale for clinical evaluation of this drug combination as well as the assessment of the extent to which BIM and/or pERK levels predict drug combination effectiveness in chemoresistant HGSOC.

  Study EGAS00001003427